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99732
GATOR Complex Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

GATOR Complex Antibody Sampler Kit #99732

Citations (0)
Western blot analysis of extracts from various cell lines using mTOR (7C10) Rabbit mAb.
Simple Western™ analysis of lysates (0.1 mg/mL) from Hela cells using mTOR (7C10) Rabbit mAb #2983. The virtual lane view (left) shows a single target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 66-440 kDa separation module.
Western blot analysis of extracts from various cell lines using Mios (D12C6) Rabbit mAb.
Immunoprecipitation of mTOR protein from MCF-7 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is mTOR (7C10) Rabbit mAb. Western blot analysis was performed using mTOR (7C10) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as the secondary antibody.
Western blot analysis of extracts from various cell lines using NPRL2 (D8K3X) Rabbit mAb.
Western blot analysis of extracts from various cell lines using WDR59 (D4Z7A) Rabbit mAb.
Western blot analysis of extracts from serum-starved NIH/3T3 cells, untreated or insulin-treated (150 nM, 5 minutes), alone or in combination with λ-phosphatase, using Phospho-mTOR (Ser2448) (D9C2) XP® Rabbit mAb (upper) or mTOR (7C10) Rabbit mAb #2983.
After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
Immunoprecipitation of Mios from MCF7 cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Mios (D12C6) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Mios (D12C6) Rabbit mAb.
Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Fluorescein Conjugate) #6201 (-) or SignalSilence® mTOR siRNA II (+), using mTOR (7C10) Rabbit mAb #2983 and α-Tubulin (11H10) Rabbit mAb #2125. mTOR (7C10) Rabbit mAb confirms silencing of mTOR expression, while the α-Tubulin (11H10) Rabbit mAb is used to control for loading and specificity of mTOR siRNA.
Immunoprecipitation of WDR59 from MCF7 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is WDR59 (D4Z7A) Rabbit mAb. Western blot analysis was performed using WDR59 (D4Z7A) Rabbit mAb.
Confocal immunofluorescent analysis of HeLa cells, rapamycin-treated (#9904, 10 nM for 2 hours, left), insulin-treated (150 nM for 6 minutes, middle) or insulin- and λ-phosphatase-treated (right), using Phospho-mTOR (Ser2448) (D9C2) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin. Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Immunohistochemical analysis of paraffin-embedded human breast carcinoma, showing cytoplasmic localization using mTOR (7C10) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using mTOR (7C10) Rabbit mAb in the presence of control peptide (left) or mTOR Blocking Peptide #1072 (right).
Immunohistochemical analysis of paraffin-embedded mouse brain using mTOR (7C10) Rabbit mAb.
Confocal immunofluorescent analysis of mouse embryonic fibroblast (MEF) cells using mTOR (7C10) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Flow cytometric analysis of A549 cells using mTOR (7C10) Rabbit mAb (solid line) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed line). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Mios (D12C6) Rabbit mAb 13557 20 µl
  • WB
  • IP
H M R 98 Rabbit IgG
NPRL2 (D8K3X) Rabbit mAb 37344 20 µl
  • WB
  • IP
H M R Mk 41 Rabbit IgG
WDR59 (D4Z7A) Rabbit mAb 53385 20 µl
  • WB
  • IP
H Mk 110 Rabbit IgG
mTOR (7C10) Rabbit mAb 2983 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
H M R Mk 289 Rabbit IgG
Phospho-mTOR (Ser2448) (D9C2) XP® Rabbit mAb 5536 20 µl
  • WB
  • IP
  • IF
H M R Mk 289 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

Product Description

The GATOR Complex Antibody Sampler Kit provides an economical means of detecting select components of the GATOR complex, mTOR and phospho-mTOR (Ser2448). The kit contains enough primary antibodies to perform at least two western blot experiments per antibody.

Specificity / Sensitivity

Mios (D12C6) Rabbit mAb recognizes endogenous levels of total Mios protein. NPRL2 (D8K3X) Rabbit mAb recognizes endogenous levels of total NPRL2 protein. WDR59 (D4Z7A) Rabbit mAb recognizes endogenous levels of total WDR59 protein. mTOR (7C10) Rabbit mAb detects endogenous levels of total mTOR protein. Phospho-mTOR (Ser2448) (D9C2) XP® Rabbit mAb detects endogenous levels of mTOR protein only when phosphorylated at Ser2448.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu730 of human Mios protein. Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala211 of human NPRL2 protein. Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding His356 of human WDR59 protein. Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser2481 of human mTOR. Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser2448 of human mTOR protein.

Background

The mTORC1 kinase complex plays a critical role in cell growth regulation (1,2). mTORC1 activity is modulated by energy levels, growth factors, and amino acids (3,4). Four related GTPases (RagA, RagB, RagC, and RagD) interact with raptor in mTORC1, which is necessary and sufficient for mTORC1 activation in response to amino acid signals (1,2). The GAP Activity Towards Rags (GATOR) complex interacts with Rag GTPases and is made up of a pair of protein subcomplexes (5). The GATOR1 subcomplex includes the proteins DEPDC5, Nprl2 and Nprl3, and is a RagA and RagB GTPase-activating protein (GAP) that negatively regulates mTORC1 signaling. Conversely, the GATOR2 subcomplex (including Mios, WDR24, WDR59, Seh1L and Sec13 proteins) is a positive regulator of mTORC1 signaling (5).
The mammalian target of rapamycin (mTOR, FRAP, RAFT) is a Ser/Thr protein kinase (6-8) that functions as an ATP and amino acid sensor to balance nutrient availability and cell growth (9,10). When sufficient nutrients are available, mTOR responds to a phosphatidic acid-mediated signal to transmit a positive signal to p70 S6 kinase and participate in the inactivation of the eIF4E inhibitor, 4E-BP1 (11). These events result in the translation of specific mRNA subpopulations. mTOR is phosphorylated at Ser2448 via the PI3 kinase/Akt signaling pathway and autophosphorylated at Ser2481 (12,13).

Pathways

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Limited Uses

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For Research Use Only. Not for Use in Diagnostic Procedures.
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U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
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